The present invention relates to the generation, by recombinant DNA methods, of novel recombinant immunoglobulins specific for the human FB5 (endosialin) cancer antigen. The invention also discloses methods for the production of these recombinant antibodies, for the diagnosis and treatment of certain human cancers.
Transformation of a normal cell to a malignant cell is often accompanied by a change in the expression of cell surface antigens. These different phenotypes can be detected using monoclonal antibodies specific for such antigens. In this way, different cancer cells can be detected and characterized (Lloyd, K. O. (1983) "Human Tumour Antigens: Detection and Characterization with Monoclonal Antibodies" in R. B. Herberman, ed., Basic and Clinical Tumour Immunology, pp 159-214, Martinus Nijhoff, Boston). These cell surface antigens are appropriate targets for tumour immunotherapy and diagnosis.
Of particular value for the diagnosis and therapy of a broad range of cancers would be the identification of an antigen associated with a broad range of cancers. Tumour stromas are potential sites for the location of such antigens. One such antigen (F19) is expressed on the surface of reactive stromal fibroblasts associated with &gt;90% of epithelial cancers (Garin-Chesa, P. et al. (1990) Proc. Natl Acad. Sci. 87, 7235-7239; Rettig, W. J. et al. (1988) Proc. Natl Acad. Sci. 85, 3110-3114). In clinical trials, a monoclonal antibody specific for the F19 antigen accumulated at tumour sites successfully locating hepatic metastases from colorectal carcinomas (Welt, S. et al. (1992) Proc. Am. Assoc. Cancer Res. 33, 319). This illustrates the diagnostic potential of monoclonal antibodies specific for tumour stromal antigens. Another tumour stromal antigen (FB5 or `endosialin`) has been identified and partially characterized (Rettig, W. J. et al. (1992) Proc. Natl Acad. Sci. 89, 10832-10836). A murine monoclonal antibody (mAbFB5) has been raised against the FB5 antigen. This antibody has been used to show that the FB5 antigen is expressed on the luminal surface of vascular endothelial cells of a wide range of malignant tumours. Specifically, in immunohistochemical analyses of vascular endothelial cells of human tumours, FB5 expression was found in 26 of 36 carcinomas, 18 of 25 neuroectodermal tumours and 41 of 61 sarcomas. In contrast, it could not be detected in any of a wide range of normal adult tissues including tissues of the following organ systems: breast, cardiovascular, connective tissues, digestive tract, endocrine, haematopoietic, lymphoid, reproductive, skin and urinary. Similary, FB5 was not expressed in cultured human malignant cells (excepting a subset of sarcomas), and stromal fibroblasts of only a small proportion of epithelial cancers exhibited FB5 expression.
The specificity of the FB5 murine antibody makes it a powerful tool for the detection of human cancers in vitro. For a number of reasons the location of the FB5 antigen on the luminal surface of tumour vascular endothelial cells makes the antigen an ideal target for tumour immunotherapy and diagnosis in vivo. Firstly, a wide range of cancer types may be diagnosed and treated by the FB5 antibody (or antibody conjugate). Secondly, the endothelial cell surface is readily accessible to antibodies that are circulating in the blood stream. Thirdly, antibody-targeted destruction of tumour blood vessels could lead to widespread necrosis in solid tumours. Finally, on binding to FB5-expressing cells the mAbFB5 is rapidly internalized raising the possibility that the antibody could be used for the specific delivery of cytoxic agents for the destruction of tumour blood vessels. However, the in vivo use of murine antibodies as agents for the diagnosis and treatment of human diseases in severely curtailed by a number of factors. Specifically, the human body recognises murine antibodies as foreign. This recognition of the murine antibodies can elicit a human anti-mouse antibody (HAMA) response (Schroff, R. et al. (1985) Cancer Res. 45, 879-885) which results in rapid clearance of the antibody from the circulation. Furthermore, the Fc portion of a murine antibody is not as efficacious as the human Fc at stimulating human complement or cell-mediated cytotoxicity. For the in vivo use of murine antibodies in diagnosis and therapy, these problems must be circumvented.
EP120694 (Celltech) and EP125023 (Genentech) disclose the development of `chimaeric` antibodies using recombinant DNA methods. Such antibodies comprise the variable regions from one species (eg mouse) and the constant regions from another species (eg human).
Such chimaeric antibodies would have the advantage that they retain the specificity of the murine antibody but can also stimulate human Fc dependent complement fixation and cell-mediated cytotoxicity. However, the murine variable regions can still elicit a HAMA response (Bruggemann, M. et al. (1989) J. Exp. Med. 170, 2153-2157) thereby limiting the value of chimaeric antibodies as diagnostic and therapeutic agents.
British Patent Application Number GB2188638A (Winter) discloses a process whereby recombinant antibodies can be generated by substitution of only the variable region CDRs of one antibody with those from another. Typically, this `CDR-grafting` technology has been applied to the generation of recombinant, pharmaceutical antibodies consisting of murine CDRs, human variable region frameworks and human constant regions (eg Riechmann, L. et al, (1988) Nature, 332, 323-327). Such `reshaped` or `humanized` antibodies have less murine content than chimaeric antibodies and retain the human constant regions necessary for the stimulation of human Fc dependent effector functions. In consequence, CDR grafted antibodies are less likely than chimaeric antibodies to evoke a HAMA response when administered to humans, their half-life in circulation should approach that of natural human antibodies and their diagnostic and therapeutic value is enhanced.
In practice, for the generation of efficacious humanized antibodies retaining the specificity of the original murine antibody, it is not usually sufficient simply to substitute CDRs. In addition there is a requirement for the inclusion of a small number of critical murine antibody residues in the human variable region. The identity of these residues depends on the structure of both the original murine antibody and the acceptor human antibody. British Patent Application Number 9019812.8 discloses a method for identifying a minimal number of substitutions of foreign residues sufficient to promote efficacious antigen binding.
The present invention provides novel, humanized monoclonal antibodies specific for the human FB5 cancer antigen. This has been achieved by the conversion of the murine FB5 monoclonal antibody to humanized antibodies by utilising CDR-grafting technologies. The invention also provides methods for the production of these humanized antibodies to be used in the diagnosis and treatment of certain human cancers. Prior to the work of the inventors, it was not known that FB5 or any other non-human antibody specific for the the FB5 antigen could be humanized so as to retain useful binding specificity.